case study

1. Background Information
Protein synthesis is a crucial process in our bodies that determines and builds the type and
number of proteins that we need to function properly. Problems in this system can lead to
difficulties in our body's functions. For instance, a mutation in eIF2 results in dysregulation of
protein synthesis leading to a rare X-linked intellectual disability known as MEHMO syndrome
(Young-Baird et al., 2020). The integrated stress response (ISR) within cells can be activated by
difficulties in protein synthesis. A specific gene, EIF2S3, has been linked to MEHMO syndrome
which causes intellectual disability, epilepsy, hypogenitalism, microcephaly, and obesity
(Skopkova et al., 2017). EIF2S3 is involved in the first steps of protein synthesis and is vital for
selecting the starting point for making a protein. In MEHMO syndrome, mutations in EIF2S3
prevent it from functioning correctly, which triggers a cellular stress response that inhibits protein
production. This response also increases the production of certain stress-related proteins. Five
different mutations in EIF2S3 have been linked to MEHMO syndrome, and some of these
mutations affect the binding of essential components in the protein synthesis process.
Among these are the mutations eIF2g-I222T, which affects eIF2b binding to eIF2g, and eIF2g-
I258M, which affects Met-tRNAi Met binding to eIF2. Moreover, hypopituitarism and glucose
dysregulation are brought on by the eIF2g-P432S mutation (Young-Baird et al., 2020). The
translation of GCN4, the yeast ortholog of ATF4, is derepressed in yeast models containing these
three mutations, indicating that the MEHMO syndrome mutations activate the ISR (Young-Baird
et al., 2020). Despite being genetically connected to MEHMO syndrome, the eIF2g-S108R
mutation has not been linked to any biochemical defects. A 4-nucleotide deletion located close to
the 30 end of the EIF2S3 coding region is present in the sixth mutation associated with MEHMO
syndrome, known as eIF2g-I465Sfs*4, which alters the C-terminal amino acid sequence of eIF2g
from G462VTIKPTVDDD* to G462VTSQQ*. In a yeast model, the eIF2g-fs mutation suppressed
GCN4 expression. It is unknown, though, how this mutation affects eIF2 function and results in
MEHMO syndrome phenotypes. Knockdown of EIF2S3 in zebrafish was found to phenocopy the
microcephaly symptom of MEHMO syndrome(Young-Baird et al., 2020).
Applied Biochemistry:
Case Study on “Suppression of MEHMO
Syndrome Mutation in eIF2 by Small
Molecule ISRIB”
Authors: Sara K. Young-Baird, Maı´ra Bertolessi Lourenco, Megan K.
Elder, Eric Klann, Stefan Liebau, Thomas E. Dever

2. Figure 1. The process of the Integrated
stress response (ISR), adapted from (Costa-
Mattioli et al., 2020).
The ISR balances bodily functions needed
to function and is triggered by specific states
such as misfolding of proteins in the
endoplasmic reticulum as well as viral
infections (Dalton et al., 2012). As shown in
Fig. 1 the ISR sensor kinases phosphorylate
eIF2, which isolates inactive eIF2.eIF2-P,
reducing the amount of eIF2 movement in
the cell resulting in the ternary complex
concentration being decreased. (Costa-
Mattioli et al., 2020). Researchers have
found a small-molecule ISR inhibitor, called
ISRIB (integrated stress response inhibitor).
This drug was discovered to restore
translation when P-eIF2a is present,
achieved by promoting the formation of
more active eIF2B (Rabouw et al., 2019).
The report discusses that the drug ISRIB,
which is an activator of eIF2B has been shown to rescue cell growth, ISR induction, and neuronal
differentiation phenotypes associated with the eIF2g-fs mutation it may be a potential treatment
for this disorder that affects cellular protein synthesis (Young-Baird et al., 2020).
Biochemical Methods
For the investigation of the effects of ISRIB on MEHMO syndrome mutation, keratinocytes were
taken from a patient with the eIF2-fs C-terminal mutation as well as a singular ex-pressing wild-
type (WT) eIF2 to create induced pluripotent stem cells(iPSCs) (Young-Barid et al., 2020). As
demonstrated in Fig. 2B, puromycin was used in actively growing cells to measure protein
synthesis and determine the speed at which the iPCS were multiplying to compare the iPCs from
a MEHMO expressing patient and the WT eIF2 cell lines. This method was applied as
puromycin is a natural aminonucleoside antibiotic, that can terminate translation prematurely by
inhibiting protein synthesis by ribosome-catalysed inclusion into the C-terminus of elongating
nascent chains. Although its primary application is as a selection marker for cell lines that have
been genetically modified to express a resistance transgene, it can also be used as a probe for
protein synthesis in a wide range of model systems, including intact cultured cells, whole
animals, and purified ribosomes. It is commonly used in experiments relating to protein synthesis
due to the fact it can adapt to certain chemical substitutions and changes without significantly
reducing activity, a wide range of puromycin-based reagents with extra functionality, like
fluorophores for fluorescence microscopy detection or biotin for affinity purification, can be
created. These tools, together with anti-puromycin antibodies, have been essential in helping us
understand how protein synthesis is regulated and dysregulated in both healthy and diseased
processes, such as immunological response and neurological function (Aviner et al., 2020). This
method was successful in showing an approximate 38% reduction in total protein synthesis in the
eIF2 iPCs whereas, the MEHMO iPCS had a significantly lower growth time, thus proving that
cell proliferation and overall protein synthesis was hindered by the mutation eIF2 greatly.
An alternative to puromycin labelling called stable isotope labelling by amino acids in cell culture
(SILAC) can be used to measure rates of protein synthesis as it is highly accurate. It entails
growing cells in a medium enhanced with amino acids that either contain heavy or normal stable
isotopes. Protein synthesis is the metabolic process by which the amino acids are integrated into
the cell's proteins. The mass difference brought about by the differential labelling makes it simple

3. to identify the source of the sample when the combined light and heavy isotope-labelled proteins
are subjected to mass spectrometry analysis. The intensities of peptides are used to calculate
the relative abundance of the proteins. Furthermore, this approach has the advantage of lower
quantitative mistakes as the samples are combined early in the experiment and the proteins and
peptides can be changed without chemical reactions (Zhang et al., 2009).
Figure 2. Results of
experiments used to
investigate eIF2g-fs
Mutation on global
translation and
expression of
important ISR factors
taken from (Young-
Baird et al., 2020).
Protein separation
and identification in
research frequently
use western blotting
as this method uses gel electrophoresis to separate a mixture of proteins according to their
molecular weight. These findings are then put on a membrane, where each protein is
represented by a band. After that, labelled antibodies particular to the target protein are
incubated on the membrane. Only the bound antibody to the target protein remains after the
unbound antibody is removed by washing. The film is then developed to identify the attached
antibodies. There should only be one band visible because the antibodies exclusively bind to the
desired protein. A standard can be used to determine the amount of protein present because the
band's thickness reflects the amount of protein present (Mahmood et al., 2012). As represented
in (Figure 2C) the western blot analysis showed elevated levels of key ISR members ATF4,
CHOP, and GADD34 in the patient-derived cell lines, which is consistent with the idea that the
eIF2g-fs mutation impairs eIF2 function. The eIF2g-fs mutation is causing the ISR independently
of eIF2a phosphorylation, as indicated by the fact that no notable differences in eIF2a-P levels
were seen between the WT and iPSCs. Since ATF4, CHOP, or GADD34 levels were not
increased by overexpressing eIF2g in WT iPSCs (Figure 2C), it is more likely that decreased
eIF2 activity in the eIF2g-fs mutant cells is the cause of the induction of the ISR rather than
altered eIF2g levels (Young-Baird et al., 2020). The procedure for western blotting is typically
simple to complete and gain viable results. However, dot blots can also be used for protein
detection and are quicker and easier to perform than western blots. They do not require
electrophoretic separation and give you swift visual confirmation of your protein expression
(Easthope 2022) whereas, western blots can result in weak or no bands due to many problems
that may arise such as high voltage, wrong concentrations, improper transfer, or air bubbles in
the gel (Mahmood et al., 2012).
The ISR translational programme was proven to be induced in the mutant iPSCs using polysome
analysis and reporter assays. In the eIF2g-fs iPSCs (Figure 2D), 59.0% ± 1.1% of ATF4 mRNA
was associated with polysomes, indicating enhanced translation, whereas in the WT iPSCs, the
majority of ATF4 mRNA was associated with monosomes and small polysomes (Young-Baird et
al., 2020).

4. Figure 4 Luciferase assays performed on ATF4, CHOP,
GADD34mRNAs, adapted from (Young-Baird et al., 2020)
A flexible and sensitive way to test several targets in high-
throughput drug screening programmes is through reporter gene
assays. A signal can be chosen from a range of reporter genes that
are compatible with assays, available detection equipment, the
biological system being used, and sensitivity. Promoters can be
selectively triggered to cause the production of a gene, or they can
be activated by a variety of metabolic pathways (Liu et al., 2009).
This method was able to show increased translation in the eIF2g-fs
iPSCs (Young-Baird et al., 2020). The enzyme luciferase is utilised
by a variety of natural species, most notably the firefly, for
bioluminescence (Carter et al., 2015). Enhanced green fluorescent
protein (EGFP) has been found to be a better alternative to
luciferase as a control reporter as it can be measured in vitro and
provide more consistent observations. EGFP compared to luciferase
was shown to handle more samples in less time and was not
dependent on plasmid concentration (Vesuna et al., 2005).

5. Figure 3. Immunofluorescent images, dendrite and branching quantification and sholl analysis
demonstrating the effects of ISRIB on neuronal differentiation defects adapted from (Young-Baird
et al., 2020).
Dendritic MAP2 immunofluorescence indicated that the WT iPSCs easily differentiated into
neurons, as seen in Figure 3A. Crucially, developing the mutant iPSC-derived neurons in the
presence of ISRIB preserved the dendritic branching characteristics, corroborating the idea that
eIF2 function is essential for strong neuronal differentiation (Young-Baird et al., 2020). The
method known as immunofluorescence (IF) was used as it makes it possible to see almost all the
constituents in any type of tissue or cell. The utilisation of certain antibody combinations that
have been fluorophore-tagged allows for this extensive capability (Im et al.,2020). In terms of
signal amplification, targeted specificity, resolution, and analytical capabilities, IF has intrinsic
advantages (Im et al.,2020). On the other hand there are some disadvantages to using IF such
as photobleaching, the photochemical breakdown of the fluorochrome. It can be avoided by
using more durable fluorochromes that are less susceptible to photobleaching, or by lowering the
light exposure intensity or duration. Furthermore, immunofluorescence is typically restricted to
fixed cells because antibodies are unable to penetrate the cell membrane (Allaire et al., 2015).
A new, developing alternative called digital spatial profiling (DSP) can be used. Formalin-fixed,
paraffin-embedded (FFPE) samples can be used to do multiplex spatial profiling of proteins or
RNAs. This methodology mainly consists of oligonucleotide tags for proteins or RNAs, which are
then linked to affinity reagents via a photocleavable linker (PL), and PL is projected onto the

6. tissue sample to release oligonucleotides in any spatial pattern across a region of interest. Using
the antibody readout, DSP can achieve single-cell sensitivity inside a ROI and identify RNA to
specific mRNAs. Compared to immunofluorescent imaging, DSP gives higher resolution imaging
and has a simpler methodology (Merritt et al., 2020).
Sholl analysis has been a crucial method in the study of dendritic anatomy and a crucial indicator
of dendritic complexity. In addition, Sholl analysis is derived by measuring the amount of dendritic
branches at a specific distance from the soma. Its uses range from assessing the structural
alterations brought on by pathologies to projecting the anticipated number of anatomical synaptic
connections (Bird et al., 2019). As represented in (Figure 3B) the eIF2g-fs neurons showed a
little reduction in the amount of dendrites in every cell, but dendritic branching showed more
significant alterations. Sholl analysis disclosed that the mutation significantly altered the intricacy
of the dendritic arbour in neurons taken from mutant iPSCs in comparison to the controls. The
number of branches per dendrite was greatly reduced in the mutant neurons (Figure 3C) (Young-
Baird et al., 2020). An experiment to quantify changes in growth of mammary glands in rats used
sholl analysis to work out the complexity of branching in rat mammary glands. The results of this
experiment showed that sholl analysis has high sensitivities as it was able to show significant
differences in branching densities (Stanko et al., 2014).
Work and Impact
The ethical applications of the small molecule ISRIB should be considered as it was not designed
to treat MEHMO syndrome and its symptoms. This therapeutic has multiple uses including age-
induced memory deficits, dendritic spine loss and reversal of age-induced immune responses
(Krukowski et al., 2020). Dendritic spine density following ISRIB treatment in aged mice was
measured with fluorescently labelled excitatory neurons to see if ISRIB might influence age-
induced synaptic structural alterations. Reduced dendritic spine density in the hippocampal
region of aged mice is associated with a decrease in cognitive function. The results of this
experiment showed that older Thy1-YFP-expressing mice were given ISRIB therapy along with
two days of behavioural training. After the animals were killed on Day 2, their brains were
removed and used in confocal microscope imaging and objective analysis to quantify the density
of dendritic spines in the hippocampus. It was found that older Thy1-YFP animals had
significantly lower dendritic spine densities than younger mice and when compared to age-
matched vehicle-treated mice, ISRIB therapy dramatically increased the number of spines. When
considered collectively, these findings show that treatment of ISRIB enhances the structure and
function of neurons in aged mice (Krukowski et al., 2020).
Moreover, human clinical trials are not easily possible as MEHMO is a rare condition affecting
less than 1000 people in the USA (NIH GARD), therefore testing the drug ISRIB on patients with
MEHMO syndrome is needed to analyse the side effects and reactions. The symptoms that
characterise MEHMO syndrome also include hypopituitarism and glucose dysregulation in some
patients (Young-Baird et al., 2020), which is not specific and more research into the EIF2S3
mutation can help clarify these, as some symptoms may be believed to be minor and are likely to
be discarded which can result in later detection and higher fatality risk. As part of the 9 diseases
neonatal screening tests for, MEHMO syndrome should be one of them as if you have the
condition life expectancy is less than 2 years. Furthermore, there is research comparing the
mutations in eIF2B that can cause leukoencephalopathy with white vanishing matter as its
symptoms are like MEHMO symptoms. To distinguish between patient phenotypes of eIF2B and
eIF2 mutations, investigating further into the molecular differences can assist in advancing
possible studies into the rare x-linked intellectual disability (Young-Baird et al., 2018).
Young-Baird et al., 2020 revealed that improved experiments can be performed to provide higher
accurate results by carrying out experiments such as dot blots, or EGFP for increased sensitivity
and precision. These can also provide more research and advancements into MEHMO
syndrome.

7. Word count: 2,520 including figures, excluding references.
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